We are continuing with the study of nucleosome assembly by investigating whether the subunits move, once deposited. This will be performed by two methods. First, the proteins associated with a given DNA sequence will be radiolabeled and, at subsequent times, chromatin will be isolated again to test whether labeled proteins have remained associated with that region of DNA or have randomized. This will be performed in synchronous Physarum polycephalum in which the replication of a given DNA sequence can be accurately timed. The other method for measuring nucleosome movement is to introduce density-labeled amino acids into nucleosomes. Oligonucleosomes of a specific size, e.g., tetramers, will be banded in metrizamide to measure the rate of density shift which should occur when light nucleosomes become interspersed with heavy ones. The activity of DNA replication activities on chromatin versus DNA templates will be investigated in vitro. The ability of DNA polymerase and DNA ligase to fill in and seal DNA associated with nucleosomes will be compared to incompletely replicated products obtained from nuclei allowed to replicate in vitro in order to study topographical relationships between the chromatin substrate and replicative enzymes.